We have used cultured long-term lymphoid cell lines from patients with cystathioninuria to show immunochemically that in the B6-unresponsive form cystathionase either is lacking or is so altered as to lose both catalytic activity and antigenic identity; in B6-responsive cases, there is present an inactive but antigenically cross-reactive protein, which is activated in vitro by added pyridoxal phosphate. We now have additional B6-responsive cases and propose application of these immunochemical studies to determine whether other mutations exist. We also will compare the kinetic and physical properties of the normal and abnormal enzymes, measure turnover rates, and study complementation in cellular hybrid. Attempts will be made to include cystathionase in unresponsive cells, as can be accomplished in fetal liver. Immunochemical delineation of cystathionine synthase from cultured cells of homocystinuric patients is a primary goal of our studies; normal synthase is being purified for the preparation of antibody. We recently have found a qualitative difference between normal and abnormal synthase, in both liver and cultured cells: the normal enzyme activated by brief heating at 55 degrees, the abnormal enzyme is not. Activation is observed in biopsied liver from patients only during B6 treatment. We propose extension of these studies to cultured cells from other affected families, particularly to the four generations of one family in which an allele for synthase deficiency is segregating. Evidence is accumulating that cystathionine synthase is a multimeric protein; thermostability studies, as well as complementation analysis with hybrids, will be utilized to relate the multimeric nature of the enzyme to the observed genetic heterogeneity. We also are studying deficiency of methionine adenosyltransferase in persistent hypermethioninemia, first described from this laboratory. Although deficient in liver, the enzyme is present in normal amounts in cultured cells and in erythrocytes, suggesting that isozymes exist which are under separate genetic control. We propose to explore this possibility.